Detection of nanometer-sized particles in living cells using modern fluorescence fluctuation methods

Nanosized materials are increasingly used in medicine and biotechnology but originate also from various aerosol sources. A detailed understanding of their interaction with cells is a prerequisite for specific applications and appraisal of hazardous effects. Fluorescence fluctuation methods are applied to follow the time-course of the translocation and distribution of fluorescent 20 nm polystyrene nanoparticles with negative surface charges in HeLa cells under almost physiological conditions. The experimental results demonstrate that singular particles enter the cell without significant contribution by endocytotic mechanisms and are distributed within the cytoplasm. Subsequently aggregation is observed, which can be blocked by cytotoxins, like Genistein and Cytochalasin B, interfering with cellular uptake processes. The observed non-active uptake is due to non-specific interactions with the cell surface and could be responsible for distribution of nanometer-sized materials in tissue.     

High sequence coverage by in-capillary proteolysis of native proteins and simultaneous analysis of the resulting peptides by nanoelectrospray ionization-mass spectrometry and tandem mass spectrometry

Losses of proteolytic peptides during extraction and/or purification procedures succeeding in-gel or in-solution digests of proteins frequently occur in the course of protein identification investigations. In order to overcome this disadvantage, the method of in-capillary digest was developed: native proteins were incubated in the presence of endoproteases in the electrospray capillary and the resulting peptides were analyzed by nanoelectrospray-mass spectrometry during the ongoing proteolysis. In-capillary digest of apomyglobin by use of trypsin in a molar ratio of 25:1 yielded complete degradation already after 15 min. The sequence coverage based on formation of molecular ions was 100% and peptide ions could be fragmented by collision-induced dissociation and sequenced. When myoglobin was incubated in the electrospray capillary with trypsin in a molar ratio of 500:1, a clear shift from molecular ions and miscleaved peptide ions to the expected final tryptic peptide ions was observed over a 2 h period. The peptide spectra obtained from tryptic in-capillary proteolysis of bovine serum albumin and apotransferrin, respectively, gave rise to sequence coverages of more than 40% for both proteins. The data obtained from the peptide maps as well as from collision-induced dissociation (CID) of selected peptides were more than sufficient for protein identification by database searches. An elephant milk protein preparation was used to demonstrate the application of in-capillary proteolysis on protein mixtures. Tryptic digest, simultaneous analysis of the proteolytic peptides by use of CID, and subsequent sequencing allowed the identification of lactoferrin, alphas1-casein, beta-casein, delta-casein, and kappa-casein by homology search.     

The soluble NAD+-Reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH

The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H(2). We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).     

Crystal structure of the Yersinia enterocolitica type III secretion chaperone SycT

Several Gram-negative pathogens deploy type III secretion systems (TTSSs) as molecular syringes to inject effector proteins into host cells. Prior to secretion, some of these effectors are accompanied by specific type III secretion chaperones. The Yersinia enterocolitica TTSS chaperone SycT escorts the effector YopT, a cysteine protease that inactivates the small GTPase RhoA of targeted host cells. We solved the crystal structure of SycT at 2.5 angstroms resolution. Despite limited sequence similarity among TTSS chaperones, the SycT structure revealed a global fold similar to that exhibited by other structurally solved TTSS chaperones. The dimerization domain of SycT, however, differed from that of all other known TTSS chaperone structures. Thus, the dimerization domain of TTSS chaperones does not likely serve as a general recognition pattern for downstream processing of effector/chaperone complexes. Yersinia Yop effectors are bound to their specific Syc chaperones close to the Yop N termini, distinct from their catalytic domains. Here, we showed that the catalytically inactive YopT(C139S) is reduced in its ability to bind SycT, suggesting an ancillary interaction between YopT and SycT. This interaction could maintain the protease inactive prior to secretion or could influence the secretion competence and folding of YopT.     

Oxytetracycline inactivation by putative reactive oxygen species released to nutrient medium of Helianthus annuus hairy root cultures

When subjected to stress, plants produce reactive oxygen species (ROS) as a part of the defense response. The oxidative response is also used to degrade organic pollutants. Hairy roots of Helianthus annuus (sunflower) are shown to oxidize oxytetracycline (OTC) through the action of the ROS released to the nutrient medium by the hairy root cultures. Methyl jasmonate (MeJA) elicits ROS formation in the hairy root cultures. The activities of the antioxidant enzymes, ascorbate peroxidase (APX), catalase (CAT), and guaiacol peroxidase (GPX), are reported for hairy root cultures treated with increasing concentrations of MeJA. A bioassay using Enterococcus hirae as the test microorganism demonstrates the root-catalyzed oxidation process results in conversion of OTC into product(s) devoid of antibiotic activity. Direct evidence for putative ROS oxidation of OTC is obtained by mass spectrometry (MS) and HPLC/MS showing first quinone formation followed possibly by ring cleavage, which disrupts UV absorption and destroys antibiotic activity.     

Hierarchical phosphorylation of the TNF-alpha receptor, TNF-R1, by p42Mapk/Erk at basic Pro-directed kinase sites

Phosphorylation of the TNF-alpha receptor TNF-R1 has been shown to differentially regulate receptor signaling and function and promote changes in its subcellular localization. Previous studies have shown that p42(mapk/erk2) phosphorylates Ser and Thr residues (T236, S240, S244, and S270) in the membrane proximal region of TNF-R1 and that mutation of these residues to Glu and Asp residues (TNF-R1.4D/E) mimics the effect of phosphorylation on receptor signaling and localization. In the present study, we investigated whether the initial phosphorylation of these residues by p42(mapk/erk2) promotes hierarchical phosphorylation of additional sites within the cytoplasmic domain of TNF-R1. This question was addressed by investigating the ability of the TNF-R1.4D/E mutant receptor to be phosphorylated in in vitro kinase assays using GST-mutant cytoplasmic domain fusion proteins as substrates and in intact cells following mutant receptor expression. In addition, we determined the location of the additional phosphorylation sites. Incubation of Sepharose bead-bound GST-TNF-R1(207)(-)(425).4D/E fusion protein with lysates containing activated p42(mapk/erk2) led to the phosphorylation of Ser and Thr residues in addition to the previously defined sites at T236, S240, S244, and S270. Deletional mutagenesis localized these residues to a stretch of 14 amino acids that encompasses three basic Pro-directed ([S/T]P) kinase consensus sequences located between residues S256 and T267. Point mutagenesis of T257, S262, and T267 to Ala residues indicated that these sites are targets of phosphorylation by p42(mapk/)(erk2). These findings support the conclusion that p42(mapk/erk2) promotes extensive phosphorylation of the membrane proximal region in a hierarchical fashion at both consensus and nonconsensus ERK-phosphorylation sites.     

Role of charged and hydrophobic residues in the oligomerization of the PYRIN domain of ASC

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein composed of two homophilic protein-protein interaction domains, a PYRIN domain (PYD) and a caspase recruitment domain. PYD-dependent oligomerization of ASC is thought to play a crucial role in formation of a molecular platform, the inflammasome, which activates caspase-1. When expressed in cells, the PYD of ASC was shown to form cytoplasmic filaments through self-association. Over 70 single point mutants were analyzed for filament formation in cells expressing the mutant proteins. The set of mutations comprised every single amino acid residue with a charged side chain (Arg, Lys, Asp, and Glu) and a large hydrophobic side chain (Ile, Leu, Met, Phe, Pro, and Val). Filament formation of the ASC PYD was prevented by mutation of Lys21, Leu25, Lys26, Pro40, Arg41, Asp48, and Asp51 of helices 2, 3, and 4. These data identify a coherent interaction surface, establishing a molecular model of PYD-PYD complexes with an important role for charge-charge interactions.     

A2A adenosine receptor induction inhibits IFN-gamma production in murine CD4+ T cells

Incubation of purified C57BL/6 murine CD4(+) T lymphocytes with anti-CD3 mAb serves as a model of TCR-mediated activation and results in increased IFN-gamma production and cell surface expression of CD25 and CD69. We demonstrate here that signaling through the TCR causes a rapid (4-h) 5-fold increase in A(2A) adenosine receptor (AR) mRNA, which is correlated with a significant increase in the efficacy of A(2A)AR-mediated cAMP accumulation in these cells. A(2A)AR activation reduces TCR-mediated production of IFN-gamma by 98% with a potency order of 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}cyclohexanecarboxylic acid methyl ester (ATL146e; EC(50) = 0.19 +/- 0.03 nM) > 4-{3-[6-amino-9-(5-cyclopropyl-carbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}piperidine-1-carboxylic acid methyl ester (ATL313; 0.43 +/- 0.06 nM) > 5'-N-ethylcarboxamidoadenosine (3.5 +/- 0.77 nM) > 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680; 7.2 +/- 1.4 nM) > N(6)-cyclohexyladenosine (110 +/- 33 nM) > 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarboxamide (390 +/- 160 nM), similar to the potency order to compete for radioligand binding to the recombinant murine A(2A)AR but not the A(3)AR. The selective A(2A)AR antagonist, 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385), inhibits the effect of ATL146e with a pA(2) of 0.34 nM and also inhibits the effects of N(6)-cyclohexyl-adenosine and 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarboxamide. In CD4(+) T cells derived from A(2A)AR(-/-) and A(2A)AR(+/-) mice, the IFN-gamma release response to ATL146e is reduced by 100 and 50%, respectively, indicative of a gene dose effect. The response of T cells to the phosphodiesterase inhibitor, 4-(3'-cyclopentyloxy-4'-methoxyphenyl)-2-pyrrolidone (rolipram), is not affected by A(2A)AR deletion. We conclude that the rapid induction of the A(2A)AR mRNA in T cells provides a mechanism for limiting T cell activation and secondary macrophage activation in inflamed tissues.     

The structures of barium hexafluorosilicate and cesium hexafluororhenate(V)

The crystal structure of CsReF₆, together with a reinvestigation of that of BaSiF₆, is reported. Both have been determined from single crystal three-dimensional X-ray diffraction data. The structure of BaSiF₆ has been found to conform to the initially assigned space group R$\text{3}$m, contrary to the suggestions of other workers. The unit cell of BaSiF₆ has the dimensiona a_hex 7.189(1), c_hex 7.015(1) Å; Z = 3. Refinement by a least squares method gave R 0.0079 and R_w 0.0077. Crystals of CsReF₆ belong to the lower symmetry rhombohedral space group R$\text{3}$. The unit cell has the dimensions a_hex 7.853(1), c_hex 8.140(1) Å; Z = 3. Refinement gave R 0.031 and R_w 0.030. The lowering of symmetry is caused by rotation of the ReF₆^? octahedra about the 3-fold axis through each Re atom, causing CsReF₆ to have the KOsF₆ structure.